Combination Therapy to Treat Hepatitis B Virus

ABSTRACT

The present invention is directed to a method for treating hepatitis B virus infection in humans comprising administering a synergistically effective amount of agents having known anti-hepatitis B virus activity in combination or alternation. Specifically, the invention is directed to a method for treating hepatitis B virus infection comprising administering FTC in combination or alternation with penciclovir, famciclovir or Bis-POM-PMEA. Additionally, the invention is directed to a method for treating hepatitis B virus infection comprising administering L-FMAU in combination or alternation with DAPD, penciclovir or Bis-POM-PMEA. The invention is further directed to a method for treating hepatitis B virus infection comprising administering DAPD in combination or alternation with Bis-POM-PMEA.

This application is a divisional of U.S. patent application Ser. No.10/374,363, filed Feb. 25, 2003, which is a continuation of U.S. patentapplication Ser. No. 09/432,247, filed on Nov. 2, 1999, issued as U.S.Pat. No. 6,528,515, which claims priority under 35 U.S.C. 119(e) to U.S.Provisional Patent Application No. 60/106,664, filed on Nov. 2, 1998,and the entire contents of all of the above applications are herebyincorporated by reference.

FIELD OF THE INVENTION

This invention is in the area of methods for the treatment of hepatitisB virus (also referred to as “HBV”) that includes administering to ahost in need thereof, an effective combination of nucleosides which haveknown anti-hepatitis B activity.

BACKGROUND OF THE INVENTION

HBV is second only to tobacco as a cause of human cancer. The mechanismby which HBV induces cancer is unknown, although it is postulated thatit may directly trigger tumor development, or indirectly trigger tumordevelopment through chronic inflammation, cirrhosis, and cellregeneration associated with the infection.

Hepatitis B virus has reached epidemic levels worldwide. After a two tothree month incubation period in which the host is unaware of theinfection, HBV infection can lead to acute hepatitis and liver damage,that causes abdominal pain, jaundice, and elevated blood levels ofcertain enzymes. HBV can cause fulminant hepatitis, a rapidlyprogressive, often fatal form of the disease in which massive sectionsof the liver are destroyed.

Patients typically recover from acute hepatitis. In some patients,however, high levels of viral antigen persist in the blood for anextended, or indefinite, period, causing a chronic infection. Chronicinfections can lead to chronic persistent hepatitis. Patients infectedwith chronic persistent HBV are most common in developing countries. Bymid-1991, there were approximately 225 million chronic carriers of HBVin Asia alone, and worldwide, almost 300 million carriers. Chronicpersistent hepatitis can cause fatigue, cirrhosis of the liver, andhepatocellular carcinoma, a primary liver cancer.

In western industrialized countries, high risk groups for HBV infectioninclude those in contact with HBV carriers or their blood samples. Theepidemiology of HBV is very similar to that of acquired immunedeficiency syndrome (AIDS), which accounts for why HBV infection iscommon among patients with AIDS or AIDS related complex. However, HBV ismore contagious than HIV.

However, more recently, vaccines have also been produced through geneticengineering and are currently used widely. Unfortunately, vaccinescannot help those already infected with HBV. Daily treatments withα-interferon, a genetically engineered protein, has also shown promise,but this therapy is only successful in about one third of treatedpatients. Further, interferon cannot be given orally.

A number of synthetic nucleosides have been identified which exhibitactivity against HBV. The (−)-enantiomer of BCH-189, known as 3TC,claimed in U.S. Pat. No. 5,539,116 to Liotta, et al., has been approvedby the U.S. Food and Drug Administration for the treatment of hepatitisB. See also EPA 0 494 119 A1 filed by BioChem Pharma, Inc.

β-2-Hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane (“FTC”),claimed in U.S. Pat. Nos. 5,814,639 and 5,914,331 to Liotta, et al.,exhibits activity against HBV. See Furman, et al., “The Anti-Hepatitis BVirus Activities, Cytotoxicities, and Anabolic Profiles of the (−) and(+) Enantiomers ofcis-5-Fluoro-1-[2-(Hydroxymethyl)-1,3-oxathiolane-5-yl]Cytosine”Antimicrobial Agents and Chemotherapy, December 1992, page 2686-2692;and Cheng, et al., Journal of Biological Chemistry, Volume 267(20),13938-13942 (1992).

U.S. Pat. Nos. 5,565,438, 5,567,688 and 5,587,362 (Chu, et al.) disclosethe use of 2′-fluoro-5-methyl-β-L-arabinofuranolyluridine (L-FMAU) forthe treatment of hepatitis B and Epstein Barr virus.

U.S. Pat. No. 5,767,122 to Emory University and The University ofGeorgia Research Foundation, Inc. discloses and claims enantiomericallypure β-D-dioxolanyl nucleosides of the formula:

wherein R is NH₂, OH, Cl, or H. A method for treating HBV infectionusing a combination of DAPD and FTC is claimed in U.S. Pat. No.5,684,010 to Raymond F. Schinazi.

Penciclovir(2-amino-1,9-dihydro-9-[4-hydroxy-3-(hydroxymethyl)butyl]-6H-purin6-one; PCV) has established activity against hepatitis B. See U.S. Pat.Nos. 5,075,445 and 5,684,153.

Adefovir (9- [2-(phosphonomethoxy)ethyl] adenine, also referred to asPMEA or [[2(6-amino-9H-purin-9-yl)ethoxy]methylphosphonic acid), alsohas established activity against hepatitis B. See for example U.S. Pat.Nos. 5,641,763 and 5,142,051.

Yale University and The University of Georgia Research Foundation, Inc.disclose the use of L-FDDC (5-fluoro-3′-thia-2′,3′-dideoxycytidine) forthe treatment of hepatitis B virus in WO 92/18517.

von Janta-Lipinski et al. disclose the use of the L-enantiomers of3′-fluoro-modified β-2′-deoxyribonucleoside 5′-triphosphates for theinhibition of hepatitis B polymerases (J. Med. Chem., 1998,41,2040-2046). Specifically, the 5′-triphosphates of 3′-deoxy-3′-fluoroβ-L-thymidine β-L-FTTP), 2′,3′-dideoxy-3′-fluoro-β-L-cytidine(β-L-FdCTP), and 2′,3′ dideoxy-3′-fluoro-β-L-5-methylcytidine(β-L-FMethCTP) were disclosed as effective inhibitors of HBV DNApolymerases.

It has been recognized that drug-resistant variants of HBV can emergeafter prolonged treatment with an antiviral agent. Drug resistance mosttypically occurs by mutation of a gene that encodes for an enzyme usedin the viral lifecycle, and most typically in the case of HBV, DNApolymerase. Recently, it has been demonstrated that the efficacy of adrug against HBV infection can be augmented by administering thecompound in combination with a second, and perhaps third, antiviralcompound that induces a different mutation from that caused by theprinciple drug. Alternatively, the pharmacokinetics, biodistribution, orother parameter of the drug can be altered by such combination therapy.In general, combination therapy induces multiple simultaneous stresseson the virus.

U.S. Pat. No. 5,808,040 discloses that L-FMAU can be administered incombination with FTC, 3TC, carbovir, acyclovir, interferon, AZT, DDI(2′,3′dideoxyinosine), DDC (2′,3′-dideoxycytidine), L-DDC, L-F-DDC, andD4T.

U.S. Pat. No. 5,674,849 discloses the use of a nucleoside in combinationwith an oligonucleotide for the treatment of a viral disease.

U.S. Pat. No. 5,684,010 discloses a method for the treatment ofhepatitis B that includes administering in combination or alternation acompound of the formula:

wherein R is NH₂, OH, or Cl, with FTC, 3TC, carbovir, or interferon.

WO 98/23285 discloses a method for the treatment or prophylaxis ofhepatitis B virus infections in a human or animal patient whichcomprises administering to the patient effective or prophylactic amountsof penciclovir (or a bioprecursor thereof such as famciclovir) andalpha-interferon.

In light of the fact that hepatitis B virus has reached epidemic levelsworldwide, and has severe and often tragic effects on the infectedpatient, there remains a strong need to provide new effective treatmentsfor humans infected with the virus that have low toxicity to the host.

Therefore, it is an object of the present invention to provide newmethods for the treatment of human patients or other hosts infected withhepatitis B virus and related conditions comprising administering asynergistically effective amount of a combination of anti-HBV agents.

SUMMARY OF THE INVENTION

It has been discovered that certain combinations of agents withhepatitis B activity are synergistic, and thus can provide enhancedbenefits to the patient when administered in an effective combination oralternation dosage pattern.

In one preferred embodiment of the present invention, a method fortreating HBV infection and related conditions in humans is disclosed,comprising administering a synergistically effective amount ofβ-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3oxathiolane (FTC),preferably substantially in the form of the (−)-optical isomer, or apharmaceutically acceptable salt, ester or prodrug thereof withpenciclovir(2-amino-1,9-dihydro-9-[4-hydroxy-3-(hydroxymethyl)butyl]-6H-purin-6-one,also referred to as “PCV”). Famciclovir, or any other bioprecursor ofpenciclovir, can be used in place of penciclovir in any embodiment ofthis invention.

Another preferred embodiment of the present invention is a method fortreating HBV infection and related conditions in humans, comprisingadministering in combination or alternation a synergistically effectiveamount of β-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)1,3-oxathiolane(FTC), preferably substantially in the form of the (−)-optical isomer,or a pharmaceutically acceptable salt, ester or prodrug thereof, with9-2-(phosphonomethoxy)ethyl]adenine (PMEA, also referred to below asBis-POM-PMEA or BP-PMEA), or a pharmaceutically acceptable salt, esteror prodrug thereof, optionally in a pharmaceutically acceptable carrier.

In another preferred embodiment of the present invention, a method fortreating HBV infection and related conditions in humans is disclosed,comprising administering in combination or alternation a synergisticallyeffective amount of 2′-fluoro-5-methyl-β-L-arabinofuranolyluridine(L-FMAU), or a pharmaceutically acceptable salt, ester or prodrugthereof, with a compound of the formula:

preferablyβ-D-(2R,4R)-2-amino-9-[(2-hydroxymethyl)-1,3-dioxolan-4-yl]purine(DAPD), which is preferably administered in substantially pure form, ora pharmaceutically acceptable salt, ester or prodrug thereof, optionallyin a pharmaceutically acceptable carrier.

In yet another preferred embodiment of the present invention, a methodfor treating HBV infection and related conditions in humans isdisclosed, comprising administering a synergistically effectivecombination or alternation amount of2′-fluoro-5-methyl-β-L-arabinofuranolyluridine (L-FMAU), or apharmaceutically acceptable salt, ester or prodrug thereof, withpenciclovir, or a pharmaceutically acceptable salt, ester or prodrugthereof, optionally in a pharmaceutically acceptable carrier.

In still another preferred embodiment of the present invention, a methodfor treating HBV infection and related conditions in humans isdisclosed, comprising administering a synergistically effective amountof 2′-fluoro-5-methyl-β-L-arabinofuranolyluridine (LFMAU), or apharmaceutically acceptable salt, ester or prodrug thereof, with9-[2-(phosphonomethoxy)ethyl]adenine (PMEA), or a pharmaceuticallyacceptable salt, ester or prodrug thereof, optionally in apharmaceutically acceptable carrier.

Another preferred embodiment of the present invention comprises a methodfor treating HBV infection and related conditions in humans, comprisingadministering a synergistically effective amount of a compound of theformula:

wherein R is NH₂, OH, H, or Cl (collectively referred to herein as theDAPD compounds), preferably,β-D-(2R,4R)-2-amino-9-[(2-hydroxymethyl)-1,3-dioxolan-4-yl]purine(DAPD), which is preferably administered in substantially pure form, ora pharmaceutically acceptable salt, ester or prodrug thereof, with PMEA,or a pharmaceutically acceptable salt, ester or prodrug thereof,optionally in a pharmaceutically acceptable carrier.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “isolated enantiomer” refers to a nucleosidecomposition that includes approximately 95% to 100%, or more preferably,over 97% of a single enantiomer of that nucleoside.

The terms “substantially pure form” or substantially free of itsopposite enantiomer refers to a nucleoside composition of one enantiomerthat includes no more than about 5% of the other enantiomer, morepreferably no more than about 2%, and most preferably less than about 1%is present.

The synergistic combination of compounds or their pharmaceuticallyacceptable esters or salts, are also useful in the prevention andtreatment of HBV infections and other related conditions such asanti-HBV antibody positive and HBV-positive conditions, chronic liverinflammation caused by HBV, cirrhosis, acute hepatitis, fulminanthepatitis, chronic persistent hepatitis, and fatigue. These synergisticformulations can also be used prophylactically to prevent or retard theprogression of clinical illness in individuals who are anti-HBV antibodyor HBV antigen positive or who have been exposed to HBV.

The active compound can be converted into a pharmaceutically acceptableester by reaction with an appropriate esterifying agents, for example,an acid halide or anhydride. The compound or its pharmaceuticallyacceptable derivative can be converted into a pharmaceuticallyacceptable salt thereof in a conventional manner, for example, bytreatment with an appropriate base. The ester or salt of the compoundcan be converted into the parent compound, for example, by hydrolysis.

The term “synergistic combination” refers to a combination of drugswhich produces a synergistic effect in vivo, or alternatively in vitroas measured according to the methods described herein.

I. Active Compounds, and Physiological Acceptable Salts Thereof

The active compounds disclosed herein are therapeutic nucleosides orcyclic or acyclic nucleoside analogs with known activity againsthepatitis B. It has been discovered that certain combinations ofnucleosides provide an advantage over monotherapy, or over othercombinations. Not all combinations of the known anti-HBV drugs provide abenefit; it is often the case that drugs act antagonistically.

The active compound can be administered as any derivative that uponadministration to the recipient, is capable of providing directly orindirectly, the parent compound, or that exhibits activity itself.Nonlimiting examples are the pharmaceutically acceptable salts(alternatively referred to as “physiologically acceptable salts”), andthe 5′ and N⁴ cytosinyl or N⁶-adeninyl acylated (esterified) derivativesof the active compound (alternatively referred to as “physiologicallyactive derivatives”). In one embodiment, the acyl group is a carboxylicacid ester in which the non-carbonyl moiety of the ester group isselected from straight, branched, or cyclic alkyl or lower alkyl,alkoxyalkyl including methoxymethyl, aralkyl including benzyl,aryloxyalkyl such as phenoxymethyl, aryl including phenyl optionallysubstituted with halogen, C₁ to C₄ alkyl or C₁ to C₄ alkoxy, or is asulfonate ester such as alkyl or aralkyl sulphonyl includingmethanesulfonyl, phosphate, including but not limited to mono, di ortriphosphate ester, trityl or monomethoxytrityl, substituted benzyl,trialkylsilyl (e.g., dimethyl-5-butylsilyl) or diphenylmethylsilyl. Arylgroups in the esters optionally comprise a phenyl group.

Modifications of the active compound, and especially at the N⁴ cytosinylor N⁶ adeninyl and 5′-O positions, can affect the bioavailability andrate of metabolism of the active species, thus providing control overthe delivery of the active species. Further, the modifications canaffect that antiviral activity of the compound, in some cases increasingthe activity over the parent compound. This can easily be assessed bypreparing the derivative and testing its antiviral activity according tothe methods described herein, or other methods known to those skilled inthe art.

Prodrugs

Any of the anti-hepatitis B agents described herein can be administeredas a prodrug to increase the activity, bioavailability, stability orotherwise alter the properties of the nucleoside. A number ofhydroxyl-bound prodrug ligands are known. In general, alkylation,acylation or other lipophilic modification of the hydroxy, mono, di ortriphosphate of the nucleoside will increase the stability of thenucleotide. Examples of substituent groups that can replace one or morehydrogens on the hydroxyl or phosphate moiety are alkyl, aryl, steroids,carbohydrates, including sugars, 1,2-diacylglycerol and alcohols. Manyare described in R. Jones and N. Bischofberger, Antiviral Research, 27(1995) 1-17. Any of these can be used in combination with the disclosednucleosides to achieve a desired effect.

Nonlimiting examples of U.S. patents that disclose suitable lipophilicsubstituents that can be covalently incorporated into the nucleoside,preferably at the 5′-OH of the nucleoside or hydroxyl of the acyclicnucleoside analogs (such as PMEA or penciclovir), include U.S. Pat. No.5,149,794 (Sep. 22, 1992, Yatvin, et al.); U.S. Pat. No. 5,194,654 (Mar.16, 1993, Hostetler, et al.); U.S. Pat. No. 5,223,263 (Jun. 29, 1993,Hostetler, et al.); U.S. Pat. No. 5,256,641 (Oct. 26, 1993, Yatvin, etal.); U.S. Pat. No. 5,411,947 (May 2, 1995, Hostetler, et al.); U.S.Pat. No. 5,463,092 (Oct. 31, 1995, Hostetler, et al.); U.S. Pat. No.5,543,389 (Aug. 6, 1996, Yatvin, et al.); U.S. Pat. No. 5,543,390 (Aug.6, 1996, Yatvin, et al.); U.S. Pat. No. 5,543,391 (Aug. 6, 1996, Yatvin,et al.); and U.S. Pat. No. 5,554,728 (Sep. 10, 1996, Basava, et al.),all of which are incorporated herein by reference.

Foreign patent applications that disclose lipophilic substituents thatcan be attached to the active compounds of the present invention, orlipophilic preparations, include WO 89/02733, WO 90/00555, WO 91/16920,WO 91/18914, WO 93/00910, WO 94/26273, EP 0 350 287, EP 93917054.4, andWO 91/19721.

II. Preparation of the Active Compounds

The therapeutic nucleosides used in the synergistic compositions of thepresent invention and processes for preparing them are known in the art.

β-2-Hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane (FTC), andits enantiomers, can be prepared by the methods disclosed in U.S. Pat.Nos. 5,204,466, 5,700,937, 5,728,575 and 5,827,727, all of which areincorporated by reference.

2′-Fluoro-5-methyl-(3-L-arabinofuranolyluridine (L-FMAU) can be preparedby the methods disclosed in U.S. Pat. Nos. 5,565,438, 5,567,688 and5,587,362 to Chu, et al. All of these patents are incorporated byreference.

Methods for the preparation of the DAPD compounds, including(2R,4R)-2-amino-9[(2-hydroxymethyl)-1,3-dioxolan-4-yl]purine (DAPD) aredisclosed in U.S. Pat. Nos. 5,767,122; 5,684,010; 5,444,063, and5,179,104, all of which are incorporated by reference.

Penciclovir can be prepared by the methods disclosed in U.S. Pat. Nos.5,075,445 and 5,684,153.

PMEA can be prepared by the methods disclosed in U.S. Pat. Nos.5,641,763 and 5,142,051.

Mono, di, and triphosphate derivatives, of the active nucleosides can beprepared as 15 described according to published methods. Themonophosphate can be prepared according to the procedure of Imai, etal., J. Org. Chem., 34(6), 1547-1550 (June 1969). The diphosphate can beprepared according to the procedure of Davisson, et al., J. Org. Chem.,52(9), 17941801 (1987). The triphosphate can be prepared according tothe procedure of Hoard, et al., J. Am. Chem. Soc., 87(8), 1785-1788(1965).

III. Combination Therapy

It has been recognized that drug-resistant variants of HBV can emergeafter prolonged treatment with an antiviral agent. Drug resistance mosttypically occurs by mutation of a gene that encodes for an enzyme usedin the viral lifecycle, and most typically in the case of HBV, DNApolymerase. Recently, it has been demonstrated that the efficacy of adrug against HBV infection can be prolonged, augmented, or restored byadministering the compound in combination or alternation with a second,and perhaps third, antiviral compound that induces a different mutationfrom that caused by the principle drug. Alternatively, thepharmacokinetics, biodistribution, or other parameter of the drug can bealtered by such combination therapy. In general, combination therapyinduces multiple simultaneous stresses on the virus.

EXAMPLE 1

Test compounds: DAPD, DXG, (−)-β-FTC, L-FMAU DMVI assay controls:Untreated cells, 3TC (lamivudine), penciclovir (PCV)

Details of the assay methodology can be found in: Korba and Gerin,Antiviral Res. 19: 55-70 (1992) and Korba, Antiviral Res. 29: 49-52(1996). The antiviral evaluations were performed on six separatecultures per each of four test concentrations. All wells, in all plates,were seeded at the same density and at the same time.

Due to the inherent variations in the levels of both intracellular andextracellular HBV DNA, only depressions greater than 3.0-fold for HBVvirion DNA from the average levels for these HBV DNA forms in untreatedcells are generally considered to be statistically significant [P<0.05](Korba and Germ, Antiviral Res., 19: 55-70, 1992). Typical values for 20extracellular HBV virion DNA in untreated cells range from 80 to 150pg/ml culture medium (average of approximately 92 pg/ml).

For reference, the manner in which the hybridization analyses wereperformed for these experiments results in an equivalence ofapproximately 1.0 pg of extracellular HBV DNA/ml culture medium to 3×10⁵viral particles/ml.

Toxicity analyses were performed in order to access whether any observedantiviral effects are due to a general effect on cell viability. Themethod used was uptake of neutral red dye, a standard and widely usedassay for cell viability in a variety of virus-host systems, includingHSV and HIV. Details of the procedure are provided in the toxicity tablelegends.

Experimental Parameters

Test compounds were received as solid material at room temperature ingood package condition. Test compounds were solubilized in 100% tissueculture grade DMSO (Sigma, Corp.) at 100 mM (DAPD, FTC, L-FMAU) or 50 mM(DXG). Daily aliquots of test 10 compounds were made in individual tubesand stored at −20° C. On each day of treatment, daily aliquots of thetest compounds were suspended into culture medium at room temperature,and immediately added to the cell cultures.

For the antiviral test analyses, confluent cultures were maintained on96-well flat bottomed tissue culture plates. Two separate (replicate)plates were used for each drug treatment. A total of 3 cultures on eachplate were treated with each of the dilutions of antiviral agents (6cultures per dilution). Cultures were treated with 9 consecutive dailydoses of the test compounds. Medium was changed daily with fresh testcompounds. Only extracellular (virion) HBV DNA levels were followed.

Toxicity analyses were performed in 96-well flat bottomed tissue cultureplates. Cells for the toxicity analyses were cultured and treated withtest compounds with the same schedule and under identical cultureconditions as used for the antiviral evaluations. Each compound wastested at 4 concentrations, each in triplicate cultures. Uptake ofneutral red dye was used to determine the relative level of toxicity 24hours following the last treatment. The absorbance of internalized dyeat 510 nM (A₅₁₀) was used for the quantitative analysis. Values arepresented as a percentage of the average A₅₁₀ values (±standarddeviations) in 9 separate cultures of untreated cells maintained on thesame 96-well plate as the test compounds.

Combination treatments were conducted using the primary analysis formatexcept that 6 serial 3-fold dilutions were used for each drugcombination and a total of 8 separate cultures were used for eachdilution of the combinations. Compounds were mixed at molar ratiosdesigned to give approximately equipotent antiviral effects based on theEC₉₀ values. Three different molar ratios were used for each combinationto allow for variability in the estimates of relative potency. Thesemolar ratios were maintained throughout the dilution series. Thecorresponding monotherapies were conducted in parallel to thecombination treatments using the standard primary assay format.

For reporting purposes, the SI, EC₅₀, EC₉₀, and CC₅₀ values reported forthe combination treatments are those of the first compound listed forthe combination mixture. The concentrations and SI, EC₅₀, EC₉₀, and CC₅₀values of the second compound in the mixture can be calculated using themolar ratio designated for that particular mixture. Further details onthe design of combination analyses as conducted for this report can befound in BE Korba (1996) Antiviral Res. 29:49.

Analyses of synergism, additivity, or antagonism were determined byanalysis of the data using the CalcuSyn™ program (Biosoft, Inc.). Thisprogram evaluates drug interactions 20 by use of the widely acceptedmethod of Chou and Talalay combined with a statistically evaluationusing the Monte Carlo statistical package. The data are displayed inseveral different formats including median-effect and dose-effectsplots, isobolograms, and combination index [CI] plots with standarddeviations. For the latter analysis, a CI greater than 1.0 indicatesantagonism and a CI less than 1.0 indicates synergism.

For the toxicity analyses associated with the combination treatments,the experimental design was limited by either/or the toxicity of themore toxic compound in the mixture or the stock concentrations (e.g.related to the total volume of DMSO that could be added to the cultureswithout inducing toxicity due to DMSO and not the test compounds).

Antiviral Evaluations

ASSAY CONTROLS: Within normal variations, levels of extracellular HBV(virion) DNA remained constant in the untreated cells over the challengeperiod. The positive treatment controls, 3TC (lamivudine) [((−)β,L,2′,3′-dideoxy-3′ thiacytidine] and penciclovir [PCV] (both purchased fromMoraveck Biochemicals, La Brea, Calif.), induced significant depressionsof HBV DNA replication at the concentrations used. The activitiesobserved for 3TC in these analyses were consistent with previousexperiments where approximately 0.15 to 0.2 μM 3TC induced a 90%depression of HBV virion DNA relative to average levels in untreatedcells after 9 days of continuous treatment of 2.2.15 cells [EC₉₀] (forexample, see Korba and Boyd, Antimicrob. Agents Chemother, (1996)40:1282-1284). The activities observed for PCV in these analyses werehigher than previously reported (EC₉₀ of approximately 0.7 to 0.9 μM,Korba and Boyd, Antimicrob. Agents Chemother. (1996) 40:1282-1284).However, the preparation of PCV used for these experiments hasconsistently produced anti-HBV activities in the range reported here inseveral other independent experiments.

TEST COMPOUNDS: Test compound DAPD, FTC, DXG, and L-FMAU inducedsignificant and selective depressions in extracellular (virion) HBV DNAlevels produced by 2.2.15 cells.

The antiviral activity of DAPD was enhanced by co-treatment with FTC.The antiviral activity of DAPD was synergistic at a 3:1 or a 1:1 molarratio at all but the highest concentrations tested. As the relativeconcentration of FTC increased, the co-operative effects of the twoagents decreased. At the 1:3 molar ratio, the two agents appeared to beantagonistic.

DAPD and PCV appeared to be antagonistic at all three molar ratios andat all concentrations.

At the 1:10 and 1:1 molar ratios, DAPD and L-FMAU appeared to beantagonistic. At the 1:3 molar ratio (approximately equipotent potenciesbased on the EC₉₀'s) the interactions of the two agents were morecomplex. DAPD and L-FMAU exhibited moderately synergistic to additiveinteractions at lower concentrations which progressed to increasinglymore antagonistic interactions at higher concentrations. Subsequenttesting, however, indicated that DAPD is synergistic with L-FMAU.

The antiviral activity of L-FMAU was enhanced by co-treatment with FTC.The antiviral activity of DAPD and FTC was moderately synergistic at a3:1 or a 10:1 molar ratio at all but the highest concentrations tested.As the relative concentration of FTC increased, the cooperative effectsof the two agents decreased. At the 1:1 molar ratio, the two agentsappeared to be antagonistic.

The antiviral activity of L-FMAU was also enhanced by co-treatment withPCV. The antiviral activity of DAPD and PCV was weakly synergistic at a1:1 or a 1:3 molar ratio at all concentrations tested. As the relativeconcentration of PCV increased, the co-operative effects of the twoagents decreased. At the 1:10 molar ratio, the two agents appeared to beantagonistic.

Toxicity Evaluations

No significant toxicity (greater than 50% depression of the dye uptakelevels observed in untreated cells) was observed for 3TC, PCV, or any ofthe test compounds at the concentrations used for the antiviralevaluations.

None of the combination treatments appeared to enhance the toxicityprofiles of either agent in the different mixtures. The toxicityprofiles of some of the combination mixtures was apparently higher thanthe corresponding monotherapies since the values are reported as afactor of the concentration of the first compound listed for eachmixture. This is especially notable for the mixtures containing PCV.However, recalculation of the toxicity profiles on 10 the basis of thesecond compound (e.g. PCV) in the mixtures revealed that all of theapparent toxicities were due to the more toxic compound and that noenhanced toxicity was present in these combinations.

EXAMPLE 2

Test compounds provided: (−)-β-FTC DMVI assay controls: untreated cells,3TC (lamivudine), penciclovir (PCV)

Details of the assay methodology were as given above. Test compoundswere received as solid material at room temperature in good packagecondition. Test compounds 20 were solubilized in 100% tissue culturegrade DMSO (Sigma, Corp.) at 100 mM. Daily aliquots of test compoundswere made in individual tubes and stored at −20° C.

TEST COMPOUNDS: Test compound FTC induced significant and selectivedepressions in extracellular (virion) HBV DNA levels produced by 2.2.15cells.

The antiviral activity of FTC was enhanced by co-treatment with PCV. Theantiviral activity of the combination therapy was synergistic at allmolar ratios tested. As the relative concentration of PCV increased, thecooperative effects of the two agents decreased.

Toxicity Evaluations

No significant toxicity (greater than 50% depression of the dye uptakelevels observed in untreated cells) was observed for 3TC, PCV, FTC, orany of the combination treatments at the concentrations used for theantiviral evaluations.

None of the combination treatments appeared to enhance the toxicityprofiles of either agent in the different mixtures. The toxicityprofiles of some of the combination mixtures was apparently higher thanthe corresponding monotherapies since the values are reported as afactor of the concentration of the first compound listed for eachmixture.

EXAMPLE 3 Combination Therapy with PMEA

Test compounds provided: PMEA, (−)-β-FTC, DAPD, L-FMAU DMVI assaycontrols: Untreated cells, 3TC (lamivudine)

Details of the assay methodology were as given above. Test compounds(except for bis-POM-PMEA) were received as powdered material on dry icein good package condition and stored at −20° C. Test compoundbis-POM-PMEA was received as a 100 mM solution in DMSO. Daily aliquotsof test compounds were made in individual tubes and stored at −20° C. Oneach day of treatment, daily aliquots of the test compounds weresuspended into culture medium at room temperature, and immediately addedto the cell cultures.

TEST COMPOUNDS (PRIMARY ANALYSES): All of the test compounds inducedsignificant and selective depressions in extracellular (virion) HBV DNAlevels produced by 2.215 cells. However, the potencies of test compounds(−)-β-FTC, DAPD and L-FMAU were lower than that observed in earlieranalyses. This was most apparent for DAPD and L-FMAU.

Bis-POM-PMEA (BP-PMEA)+FTC. The mixture of BP-PMEA and FTC produced ananti-HBV activity that was moderately synergistic overall. The potencyof the mixtures increased as the relative proportion of FTC increased.However, the most favorable overall interactions occurred where theconcentration of FTC was proportionately lower. The same relative degreeof synergism was generally observed at all concentrations of the 30:1mixture. Relatively more synergistic interactions were observed at thelower concentrations of the 10:1 and 3:1 mixture and moderate to strongantagonism was observed at the highest concentrations of the 3:1mixture.

BP-PMEA+DAPD. The mixture of BP-PMEA and DAPD produced an anti-HBVactivity that was moderately to weakly synergistic at lower relativeconcentrations of DAPD and moderately to strongly antagonistic at higherrelative concentrations of DAPD. The potency of the mixtures alsodecreased as the relative proportion of DAPD increased. Relatively moresynergistic interactions were observed at the lower concentrations ofthe different mixtures.

BP-PMEA+L-FMAU. The mixture of BP-PMEA and L-FMAU produced an antiHBVactivity that was moderately synergistic at lower relativeconcentrations of L-FMAU and additive to weakly antagonistic at higherrelative concentrations of L-FMAU. The potency of the mixtures waslowest at the highest relative concentration L-FMAU (1:1 molar ratio).The most favorable overall interactions were observed at the 3:1 molarratio of the two compounds. Relatively more synergistic interactionswere observed at the lower concentrations of different mixtures.

Toxicity Evaluations

No significant toxicity (greater than 50% depression of the dye uptakelevels observed in untreated cells) was observed for 3TC, any of thetest compounds, or any of the compound mixtures at the concentrationsused for the antiviral evaluations. None of the compound mixturesappeared to significantly enhance toxicity. The patterns of toxicityobserved for the compound mixtures was similar to, and consistent with,that observed for the monotherapies.

IV. Preparation of Pharmaceutical Compositions

Humans suffering from any of the diseases described herein arising outof HBV infection, can be treated by administering to the patient aneffective amount of identified synergistic anti-HBV agents in acombination or independent dosage form for combination or alternationtherapy, optionally in a pharmaceutically acceptable carrier or diluent.The active materials can be administered by any appropriate route, forexample, orally, parenterally, intravenously, intradermally,subcutaneously, or topically, in liquid or solid form.

The active compounds are included in the pharmaceutically acceptablecarriers or diluents in amounts sufficient to deliver to a patient atherapeutically effective amount of compound to inhibit viralreplication in vivo, especially HBV replication, without causing serioustoxic effects in the patient treated. By “inhibitory amount” is meant anamount of active ingredient sufficient to exert an inhibitory effect asmeasured by, for example, an assay such as the ones described herein.

A preferred dose of the compound for all the above-mentioned conditionswill be in the range from about 1 to 50 mg/kg, preferably 1 to 20 mg/kg,of body weight per day, more generally 0.1 to about 100 mg per kilogrambody weight of the recipient per day. The effective dosage range of thepharmaceutically acceptable derivatives can be calculated based on theweight of the parent nucleoside to be delivered. If the derivativeexhibits activity in itself, the effective dosage can be estimated asabove using the weight of the derivative, or by other means known tothose skilled in the art.

The compound is conveniently administered in unit or any suitable dosageform, including but not limited to one containing 7 to 3000 mg,preferably 70 to 1400 mg of active ingredient per unit dosage form. Anoral dosage of 50-1000 mg is usually convenient, more typically 50-300mg.

Ideally the active ingredient should be administered to achieve peakplasma concentrations of the active compound of from about 0.2 to 70 μM,preferably about 1.0 to 10 μM. This may be achieved, for example, by theintravenous injection of a 0.1 to 5% solution of the active ingredient,optionally in saline, or administered as a bolus of the activeingredient.

The concentration of active compound in the drug composition will dependon absorption, inactivation, and excretion rates of the drug as well asother factors known to those of skill in the art. It is to be noted thatdosage values will also vary with the severity of the condition to bealleviated. It is to be further understood that for any particularsubject, specific dosage regimens should be adjusted over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat the concentration ranges set forth herein are exemplary only andare not intended to limit the scope or practice of the claimedcomposition. The active ingredient may be administered at once, or maybe divided into a number of smaller doses to be administered at varyingintervals of time.

A preferred mode of administration of the active compound is oral. Oralcompositions will generally include an inert diluent or an ediblecarrier. They may be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition.

The tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a bindersuch as microcrystalline cellulose, gum tragacanth or gelatin; anexcipient such as starch or lactose, a disintegrating agent such asalginic acid, Primogel, or corn starch; a lubricant such as magnesiumstearate or Sterotes; a glidant such as colloidal silicon dioxide; asweetening agent such as sucrose or saccharin; or a flavoring agent suchas peppermint, methyl salicylate, or orange flavoring. When the dosageunit form is a capsule, it can contain, in addition to material of theabove type, a liquid carrier such as a fatty oil. In addition, dosageunit forms can contain various other materials which modify the physicalform of the dosage unit, for example, coatings of sugar, shellac, orother enteric agents.

The compound can be administered as a component of an elixir,suspension, syrup, wafer, chewing gum or the like. A syrup may contain,in addition to the active compounds, sucrose as a sweetening agent andcertain preservatives, dyes and colorings and flavors.

The compound or a pharmaceutically acceptable derivative or salt thereofcan also be mixed with other active materials that do not impair thedesired action, or with materials that supplement the desired action,such as antibiotics, antifungals, antiinflammatories, proteaseinhibitors, or other nucleoside or nonnucleoside antiviral agents, asdiscussed in more detail above. Solutions or suspensions used forparenteral, intradermal, subcutaneous, or topical application caninclude the following components: a sterile diluent such as water forinjection, saline solution, fixed oils, polyethylene glycols, glycerine,propylene glycol or other synthetic solvents; antibacterial agents suchas benzyl alcohol or methyl parabens; antioxidants such as ascorbic acidor sodium bisulfite; cheating agents such as ethylenediaminetetraaceticacid; buffers such as acetates, citrates or phosphates and agents forthe adjustment of tonicity such as sodium chloride or dextrose. Theparental preparation can be enclosed in ampoules, disposable syringes ormultiple dose vials made of glass or plastic.

If administered intravenously, preferred carriers are physiologicalsaline or phosphate buffered saline (PBS).

In a preferred embodiment, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation.

Liposomal suspensions (including liposomes targeted to infected cellswith monoclonal antibodies to viral antigens) are also preferred aspharmaceutically acceptable carriers. These may he prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,81 1 (which is incorporated herein by reference inits entirety). For example, liposome formulations may be prepared bydissolving appropriate lipid(s) such as stearoyl phosphatidylethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidylcholine, and cholesterol) in an inorganic solvent that is thenevaporated, leaving behind a thin film of dried lipid on the surface ofthe container. An aqueous solution of the active compound or itsmonophosphate, diphosphate, and/or triphosphate derivatives is thenintroduced into the container. The container is then swirled by hand tofree lipid material from the sides of the container and to disperselipid aggregates, thereby forming the liposomal suspension.

This invention has been described with reference to its preferredembodiments. Variations and modifications of the invention, will beobvious to those skilled in the art from the foregoing detaileddescription of the invention. It is intended that all of thesevariations and modifications be included within the scope of thisinvention.

1. A method for the treatment of hepatitis B virus in a human comprisingadministering in combination or alternation a synergistically effectiveamount of β-2 hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane(β-FTC) or a pharmaceutically acceptable salt, ester, or prodrug thereofwith an effective amount of a second anti-hepatitis B agent selectedfrom the group consisting of famciclovir and Bis-POM-PMEA.
 2. The methodof claim 1, wherein the β-FTC is in the form of the (−)-optical isomer.3. The method of claim 1, wherein the second anti-hepatitis B agent isfamciclovir.
 4. The method of claim 1, wherein the second anti-hepatitisB agent is Bis-POM-PMEA.
 5. A pharmaceutical composition for thetreatment of hepatitis B virus in a human comprising an effective amountof β-2-hydroxymethyl-5 -(5-fluorocytosin-1-yl)-1,3 oxathiolane (β-FTC)or a pharmaceutically acceptable salt, ester, or prodrug thereof in asynergistic combination with an effective amount of a secondanti-hepatitis B agent selected from the group consisting of famciclovirand Bis-POM-PMEA.
 6. The composition of claim 5, wherein the β-FTC is inthe form of the (−)-optical isomer.
 7. The composition of claim 5,wherein the second anti-hepatitis B agent is famciclovir.
 8. Thecomposition of claim 5, wherein the second anti-hepatitis B agent isBis-POM-PMEA.